Markers used in this study included: D11S1986, D11S1998 (for
CRYAB); D21S2055, D21S226, and D21S1446 (for
CRYAA); and D1S1597, D1S3669, D1S1653, D1S1679, D2S1384,
D2S434, D3S2460, D3S1764, D3S1311, D13S787, D14S587, D14S592, D16S2624,
D16S539, D17S1308, D17S2196, D17S1294, D17S2193, D17S784, and D22S420
for the additional 12 loci described in autosomal dominant
cataract.
10 11 12 13 14 15 16 17 18 19 20 21 Amplification was performed in a 10-μl
reaction volume containing 50 ng of DNA, 13.4 ng of each unlabeled
primer, 1.5 mM each dNTP, and 0.08 μg
32P-labeled primer in 1.5 mM
MgCl
2 polymerase chain reaction (PCR) buffer,
with 1.2 U
Taq polymerase (Bio-Line, London, UK). After an
initial denaturation of 5 minutes at 95°C, 31 cycles were performed
at 94°C for 2 minutes, 52°C for 3 minutes, and 72°C for 1 minute,
followed by a final extension time of 7 minutes at 72°C. Samples were
mixed with 10 μl of loading buffer, denatured at 95°C for 5 minutes
and electrophoresed on a 6% denaturing polyacrylamide gel.
Single-strand conformational polymorphism (SSCP) analysis of the
CRYAA gene was performed by amplifying exons 1, 2, and 3
using the primers 5′-CTCCAGGTCCCCGTGGTACCA-3′ and
5′-GCGAGGAGAGGCCAGCACCAC-3′, 5′-CTGTCTCTGCCAACCCCAGCAG-3′ and
5′-CCCCTGTCCCACCTCTCAGTGCC-3′, 5′-GGGGAGCCAGCCGAGGCAATG-3′
and 5′-GGCAGCTTCTCTGGCATGGGG-3′, respectively. The reaction was
performed as described earlier. Samples were amplified using the
conditions described for genotyping. Samples were mixed with 10 μl of
loading buffer, denatured at 94°C for 5 minutes, and electrophoresed
on a 6% polyacrylamide-10% glycerol nondenaturing gel at 8 W for 16
hours.